The goal of this project is to develop a reliable protocol, using cryopreserved sperm, to produce, at minimum cost, the maximum number of mouse pups per male. Alternative strategies were analyzed before selecting our approach. The sequence of steps in a cryopreservation cycle, both theoretical and pragmatic, was analyzed to establish a template against which experimental variable can be tested. Likewise, the sequence to steps leading to successful fertilization was analyzed to select methods for assessment of sperm status for a hierarchal mode of detecting cryopreservation induced spermatozoal dysfunction. Final experimental design accommodates early success (ie.. luck) but assures that we will learn from initial failures. Response surface methodology will be used to simultaneously assess the effects of 3-5 levels of freezing rate, thaw rate, extender and composition of cryoprotectant on the success and reproducibility of mouse sperm cryopreservation. The results from one stage are used to plan more refined and focused assessment of a subset of the original (plus additional factors) at the next stage. This approach provides the most efficient method to establish optimal conditions for cryopreservation of sperm from multiple strains of mice. Progressive assays, chosen to follow the steps of fertilization, include: (a) preliminary screening via assay of spermatozoal motility and membrane compartments (by flow cytometry); (b) a unique assay for loose sperm-egg binding (c) sperm penetration into the zona pellucida: (d) early embryo development; and (e) final production of pups. Identification of an optimal protocol is an interactive process, requiring "yes/no" decisions, based upon experience, from assays with ill-defined cut off criteria. We (and no one else) have confidence that the "right" decision was made when "cut off" values were established. All of our data will be stored for periodic retrieval and retrospective analysis during the project period. This, we can return to our data sets (and our decisions) armed with knowledge of the overall success of that specific paradigm in retaining fertilization potential. This will allow us to learn as we evolve, and make ever better decisions, toward the successful protocol. The team assembled for this project is capable of: (a) driving an interactive program that will satisfy the stated goals of this RFA; and (b) providing a template of experimental design useful for solution of other cryobiological problems in human medicine and animal agriculture.